重组质粒pcFlaA在青石斑鱼肌肉中的表达研究  

Study on the expression of recombined plasmid pcFlaA in Epinephelus awoara

作　　者：覃映雪[1,2] 王军[1] 王世峰[1] 苏永全[1] 庄轩[1] 

机构地区：[1]厦门大学海洋系亚热带海洋研究所,厦门361005 [2]集美大学水产学院,厦门361021

出　　处：《高技术通讯》2007年第7期761-764,共4页Chinese High Technology Letters

基　　金：863计划（2003AA603011）和福建省科技重大专项（2004NJ03）资助项目.

摘　　要：在哈维氏弧菌TS-628菌株鞭毛丝蛋白FlaA基因末端加上一段编码Flag短肽的核苷酸序列作为检测标记后,将该基因克隆到真核表达载体pcDNA3.1（＋）,酶切、PCR扩增及重组质粒测序证实基因片段插入正确,将该重组质粒命名为pcFlaA.将pcFlaA以肌肉注射方式免疫青石斑鱼.免疫后第7天开始检测鞭毛丝蛋白在石斑鱼肌肉中的表达状况,之后每隔1周检测1次,共检测4次.首先采用PCR技术在DNA水平检测重组质粒转染石斑鱼肌肉细胞的情况,再以RT-PCR法在mRNA水平上检测转染质粒在鱼肌肉中的转录,最后以免疫组化染色技术在蛋白质水平上检测目的蛋白的表达.结果在DNA及mRNA水平上均可检测到目的条带,在蛋白质水平上可检测到明显阳性位点,由此证实pcFlaA可以转染石斑鱼肌肉细胞并可在其中进行表达,而且质粒在鱼体内持续表达的时间至少1个月.The FlaA gene of Vibrio harveyi, with a short nucleotide sequence encoding Flag tag as a marker, was cloned to eukaryotic expression vector pcDNA3.1 （ ＋ ）. The cloning gene was correctly inserted into the vector pcDNA3.1 （ ＋ ） confinned by restriction endonuclease digestion, PCR amplification and sequencing. The recombined plasmid was designated as pc Fla A. The experimental grouper Epinephelus awoara was immunized with pc FlaA by intramuscular injection. PCR performation was conducted to identify if the fish muscle cells were transfected with pcFlaA. The expression of pcFlaA was analyzed by RT-PCR at the level of mRNA and by immunohistochemistry at the level of protein. The results demonstrated that pcFlaA could transfect the fish muscle and keep expressing in the cells at least for one month.

关 键 词：DNA疫苗 哈维氏弧菌 青石斑鱼 

分 类 号：Q78[生物学—分子生物学]